Fazeelat Karamat Laboratoire d’Agronomie et Environnement Director of thesis : Hehn Alain

FUNCTIONAL CHARACTERIZATION OF THE FIRST PRENYLTRANSFERASE INVOLVED IN THE BIOSYNTHESIS OF COUMARINS, IMPORTANT MOLECULES IN THE DEFENSE SYSTEM OF HIGHER PLANTS

Fazeelat Karamat Laboratoire d’Agronomie et Environnement

Director of thesis : Hehn Alain Co-Director of thesis: Bourgaud Frédéric /

Olry Alexandre

Séminaire RP2E - 19/01/2012 – F. Karamat

Implication of coumarins

Self defense system of plants (Insecticides, Bactericide and Allelochemicals)

Intr

oduct

ion

O OR7

R6

R5

R8

What are coumarin?


Other implications in different industries

Pharmaceutical industry (Anti coagulant, Antitumoral, Anti-HIV, Antifungic)

Cosmetic industry ( Dye , Fragrances)

Food industry ( Flavors)

NH2HOOC HOOC HOOC

OH

Phenylalanine Cinnamic acid p-coumaroyl coA

Lignin

Flavonoid

Linear form

PAL C4H/4CL4CL / HCT / C3’H…

C2’H

O OOHO OOH

O OOH

Angular form

Umbelliferone

COCoA

Furanocoumarins

Prenyltransferase Prenyltransferase

Chlorogenic Acid…….

O OOO OO

Biosynthetic pathway of phenylpropanoid

Intr

oduct

ion


DMS Osthenol

The addition of an isoprene unit

Prenylated phenols in plants

Leguminosae EuphorbiaceaeRutaceae

MoraceaeUmbelliferae

(Park et al., 2003;Stevens et al., 2000; Riberio et al., 2008)

Prenyltransferase (PT) : enzyme that catalyze the prenylation step

PPi O ORO OR

Isoprene group polyphenol Prenylated polyphenol

What is prenylation?


Intr

oduct

ion

Anti-cancer

Anti-tyrosinase

Anti-glycosidase

Phytoalexin

Anti-poliferative activityDrug enhancement effects

Anti-oxidantBactericide

EST Sequencing

Intr

oduct

ion


Prenylated phenols in plants

Leguminosae RutaceaeUmbelliferae

SfN8DT Celery, parsley Lime, lemon, RutaAngelica

1) Identification of new candidate genes

Experi

menta

l st

rate

gy


Flavonoids

Vitamin E

Flavonoids or others?

Done by Prof Yazaki (collaboration)

Plant Heterologous expression system

Nicotiana benthamiana.

Experimental strategy

Experi

menta

l st

rate

gy

Identification of new candidate genes

Cloning of the genes in plasmidic expression vectors

Heterologous expression of the corresponding proteins


Functional characterization of proteins

N-TermORFPromotor

2) Heterologous expression system

The principle of the approach

(Voinnet et al., 2003; Kapila et al., 1997)Séminaire RP2E - 19/01/2012 – F. Karamat

Protein extraction(4 days post inoculation)

Plasmid with concerned gene

Experi

menta

l st

rate

gy

Agrobacterium

Validation of the system in two steps

1) Test with the Jellyfish Green Fluorescent Protein (GFP)

-GFP +GFP

Visible spectra

Fluorescence excitation/emission spectra


Experi

menta

l st

rate

gy

GFPPromotor N-Term


Validation of the system in two steps

2) Test with a membrane-bound-enzyme : the Sophora flavescens prenyltransferase ( a flavonoid specific prenyltransferase that has been characterized already)

N-TermSfN8DTPromotor


Infiltration of tobacco leaves

Crude membrane extracts

Ultra-turrax was used to prepare a homogeniezed mixture of fresh plant material.

Centrifugation and ultracentrifugation was done to get membrane pellets

4 days post inoculation


Experi

menta

l st

rate

gy

The heterologous expression system is functional

Reaction

Without enzyme

With enzyme (presence of 8dN)naringenin

Validation of the system in two steps2) Test with an enzyme : the Sophora flavescens prenyltransferase

Results

SfN8DTPromotor N-Term


Pri

mary

R

esu

lts

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 m in0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

uV

Test of the 6 candidate genesDMAPP GPP FPP

Xanthotoxol - - -

Bergaptol - - -

Herniarine - - -

Umbelliferone - -

6- methoxy coumarin

- - -

7- methoxy coumarin

- - -

6,7- methoxy coumarin

- - -

Coumarin - - -

7-ethoxy coumarin - - -

5-methoxypsoralen - - -

8-methoxypsoralen - - -

psoralen - - -

Isoprenic moeity

PT-Lime

O OOHO OOH

Putative PT ORF Promotor N-Term

Without enzyme With enzyme


Resu

lts

Expression of 6 candidate PTs genes

PT-Ruta

PT-Lemon PT-Angelic

PT-Parsley PT-Celery

Optimal pH=8.2 Km=24 +/-3 µM

Biochemical parameters?

Resu

lts


In agreement with already characterized prenyltransferase (Sfn8DT, SfG6DT)

O OO

Inhibition of the reaction with psoralen

Biochemical parameters?

Resu

lts


Umbelliferone

Demetylsuberosin Osthenol

Linear furanocoumarins

Angular furanocoumarins

Psoralen Angelicin

Prenyltransferase

Phenylalanine

Qua

ntit

y O

f D

MS

in n

M

Conc. Of Psoralen in µM

In this work we developed another way to screen the activity of PT enzymes using a plant transient expression system (previously it was done in yeast)

We showed a potential way to regulate the production of furanocoumarins in plants thanks to the negative feedback of psoralen

Umbelliferone


Linear furanocoumarins

Angular furanocoumarins

Psoralen Angelicin

Prenyltransferase Prenyltransferase

Concl

usi

on a

nd

pers

pect

ive

Conclusions


-

We characterized a new enzyme involved in the biosynthetic pathway of coumarins.

It is the first prenyltransferase identified in parsley, a furanocoumarin producing plant.

Phenylalanine

1) Parsley PT

In parsley - Tissue specific expression pattern of gene ( leaves, stem, roots and flower)

- Subcellular localization ( mitochondria, endoplasmic reticulum)

In Ruta graveolens :

-Stable overexpression of the gene in order to do metabolic engineering

Concl

usi

on a

nd

pers

pect

ive


Project perspective

2) The five other PT genes ?

-Enlarged metabolic screening

-Stable expression in Ruta graveolens (under way) / metabolic characterization

Umbelliferone


Prenyltransferase

Without enzyme With enzymeLinear furanocoumarins

Psoralen

O OOHO OOH

PT- parsley is bifunctional?

Fazeelat Karamat Laboratoire d’Agronomie et Environnement Director of thesis : Hehn Alain

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